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首页> 美国卫生研究院文献>World Journal of Gastroenterology >Construction and characterization of bivalent vaccine candidate expressing HspA and Mr18000 OMP from Helicobacter pylori
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Construction and characterization of bivalent vaccine candidate expressing HspA and Mr18000 OMP from Helicobacter pylori

机译：幽门螺杆菌表达HspA和Mr18000 OMP的二价疫苗候选物的构建和鉴定

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AIM: To construct a recombinant vector which can express outer membrane protein (OMP) with Mr18000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection.METHODS: The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, BamH I simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp18, digested by restrictive endonuclease enzyme Hind III and BamH I simultaneously. pET32a(+)/ HspA and Omp18 were recovered from 1% agarose gel by gel kit, and ligated with T4 ligase by BamH I digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp18 was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot.RESULTS: Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al there were 1.3% and 1.4% differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (Mr) of the expressed product was Mr 51000, Mr of protein expressed by pET32a (+) was about Mr 20000, and soluble expression product accounted for 18.96% of total bacterial protein. After purification with Ni+2-NTA agarose resins, the purification of recombinant fusion protein was about 95%. Western blot showed that recombinant fusion protein could be recognized by the patients’ serum infected with H. pylori and anti-Omp18 monoclone, suggesting that this protein had good antigenicity.CONCLUSION: The gene coding for H. pylori Mr18000 OMP and HspA was cloned and expressed successfully. The results obtained lay the foundation for development of H. pylori protein vaccine and a quick diagnostic kit.
机译：目的：构建可表达Mr18000外膜蛋白（OMP）和幽门螺杆菌（H. pylori）热休克蛋白A（HspA）在大肠杆菌BL21中表达的重组载体，探索获得疫苗的可能性。方法：通过PCR从幽门螺杆菌染色体扩增HspA的靶基因，然后同时通过限制性内切酶kpn I，BamHI将其插入原核表达载体pET32a（+）中。重组载体用于测序，然后与pET32a（+）/ Omp18一起被限制性核酸内切酶Hind III和BamHI消化。通过凝胶试剂盒从1％琼脂糖凝胶中回收pET32a（+）/ HspA和Omp18，并通过BamHI消化的粘度末端与T4连接酶连接。 pET32a（+）/ HspA / Omp18的重组质粒在IPTG的诱导下转化并在大肠杆菌BL21（DE3）中表达。结果：酶切分析和测序表明，目的基因插入到重组载体中，由891个碱基对组成，编码目的氨基酸序列为297个氨基酸残基。与Tomb等报道的GenBank相比，幽门螺杆菌的核苷酸序列和氨基酸残基分别有1.3％和1.4％的差异。 SDS-PAGE分析表明，表达产物的相对分子质量（Mr）为Mr 51000，pET32a（+）表达的蛋白质的 M r约为 M r 20000，可溶性表达产物占细菌总蛋白的18.96％。用Ni +2 -NTA琼脂糖树脂纯化后，重组融合蛋白的纯化率约为95％。 Western印迹显示重组融合蛋白可以被感染 H的患者血清识别。幽门螺杆菌和抗Omp18单克隆抗体，提示该蛋白具有良好的抗原性。结论： H的编码基因。成功克隆并表达了幽门螺杆菌M r18000 OMP和HspA。获得的结果为 H 的发展奠定了基础。 pylori 蛋白疫苗和快速诊断试剂盒。

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